The present invention relates to a novel use of sargachromenol, more particularly to a method for destroying and/or lysing keratinocytes using sargachromenol, a use for preparing an agent for lysing keratinocytes, and a composition for lysing keratinocytes.
Skin disease is caused by a variety of reasons. Because the skin, which is located at the outermost part of our body and covers the surface thereof, is always exposed to outside, skin disease may be caused by various skin irritating factors and pathogens. Further, it may be induced by hereditary factors, inflammations, benign or malignant tumors, hormones, injuries, or pathological changes such as retroplasia. Especially, damage to the stratum corneum, which is the outermost layer of the skin, may be a direct cause of skin disease.
When keratinocytes in the stratum basale of the epidermis divide through cell division, newly formed cells go upward to the outermost layer of the skin. There, the cells lose nuclei, become cornified and fall off the skin. This is called the turnover of the epidermis. That is, our skin is not maintained as it is after formed once, but is continually generated and lost. However, when the keratinocyte cell cycle is out of order because of genetic abnormalities, immunological abnormalities, infections by pathogens or mechanical or physical stimulations, a large number of problems may occur. Most skin diseases are caused by such reasons.
Apoptosis is a programmed cell death and may occur under several physiological and pathological situations. In normal cell tissues, cell proliferation and apoptosis are balanced and the number of cells in the tissue is maintained constant. However, in cancer or tumor cell tissues, the number of cells increases dynamically because apoptosis does not match the rapid cell proliferation [Raff. M. C., Nature, 356:397. 1992]. Accordingly, control of apoptosis may be a solution to the prevention or improvement of skin disease caused by hyperproliferation of keratinocytes.
Typical examples of skin disease caused by the hyperproliferation of keratinocytes include skin aging, photoaging and pigmentary diseases. Skin aging and photoaging are caused by external factors such as chronic exposure to UV associated with generation of oxygen free radicals inside the body, which result in peroxidation of the lipid components of the cell membrane and hardening of the cell membrane, thereby interfering with the supply of oxygen and nutrients into cells. As skin metabolism and replacement of skin cells are retarded because of the reasons, the stratum corneum of the skin becomes thicker and the skin experiences a lot of changes in structure and function. As a result, series of skin aging and photoaging are occurred, appearing many characteristics such as skin aging, loss of elasticity, wrinkle formation, decline of moisture holding capacity, droop and the like [Wulf H C. et al., Micron. 2004, 35, 185-91; Haake A R, et al., J. Investigative Dermatol. Symp. Proc. 1998, 3, 28-35]. In addition, pigmentation occurs, such as chloasma, dark spots, and the like [Young A R. Prog. Biophys. Mol. Biol. 2006 , 92(1), 80-5, HE. Br. J. Dermatol. 1992 , 127 Suppl. 41:48-50, Pawlowski A et al., Acta Derm. Venereol. 1991, 71(2), 113-7]. Accordingly, destroying of keratinocytes in thickened cells is effective in preventing these symptoms.
Mechanical or chemical peeling is carried out to remove keratinocytes. However, chemical peeling tends to induce irritations, inflammations, and the like. For example, chemical peeling agents such as lactic acid, glycolic acid, etc. have been used to remove fine wrinkles through facilitated removal of the horny layer. But, their use is restricted because they may cause skin irritation at specific concentration or pH. Accordingly, development of an improved agent for removing keratinocytes without skin side effects such as irritations, inflammations, and the like is needed.
In the present description, several literatures and patent publications are cited. They are specified in parentheses. The disclosure of the cited literatures and patent publications is enclosed in the present description in its entirety, and serves to describe the technical field to which the present invention belongs and the contents of the present invention more clearly.
[Disclosure]
The inventors of the present invention have been working on materials having keratinocyte removing and/or lysing activity and with little skin irritation or other side effect. As a result, we isolated and purified sargachromenol from naturally growing Sargassum sagamianum, and found out, for the first time, that the sargachromenol of the present invention exhibits a remarkable activity of inducing apoptosis of keratinocytes.
Accordingly, in an aspect, the present invention provides a method for destroying and/or lysing skin keratinocytes comprising applying sargachromenol represented by the following Chemical Formula I on the skin area where keratinocytes are hyperproliferating:

In another aspect, the present invention provides a use of the sargachromenol represented by Chemical Formula I for preparing a keratinocyte lysing agent.
In still another aspect, the present invention provides a composition for lysing keratinocytes comprising the sargachromenol represented by Chemical Formula I and a dermatologically acceptable vehicle.
The above and other aspect, objects, features and other advantages of the present invention will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings and claims.
Best Mode
Hereunder is given a more detailed description of the present invention.
The sargachromenol of the present invention can be represented by the following Chemical Formula I.

The sargachromenol of the present invention can be obtained and isolated from the nature. That is, it can be obtained from plant using the conventional method for extracting and isolating substances. The extract can be obtained by extracting using an adequate organic solvent after drying and maceration or after drying only. The resultant extract can be purified by a purification method well known to those skilled in the art to which the present invention belongs. Preferably, the sargachromenol of the present invention may be obtained and isolated from Sargassum sagamianum. 
Sargassum sagamianum is a seaweed belonging to the family Sargassaceae and is distributed along the eastern and southern coasts of Korea and the coasts of Japan. Sargachromenol extracted from Sargassum sagamianum is reported to have NGF (nerve growth factor)-dependent nerve cell growth promoting activity [Chi Kwan Tsang et al, Neuroscience, 2005]. However, there is no report on skin disease prevention or improvement activity of sargachromenol.
For the isolation and the purification of the inventive sargachromenol, first of all, an extract was prepared from Sargassum sagamianum by using water or C1-C6 organic solvent. The said organic solvent may be methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether alone or mixed together. Preferably, it may be methanol, and most preferably it may be chloroform or ethyl acetate. The said methanol extract was performed column chromatography using column filled with synthetic resin such as silica gel or activated alumina and TLC alone or together, and as a result, sargaquinoic acid was isolated. Preferably, silica gel column chromatography and octadecyl silica gel column chromatography may be used in parallel. The extract was prepared from the said sargaquinoic acid by using alcohol and chloroform solvent. The said alcohol may be methanol, ethanol, propanol, isopropanol, butanol and the like and it may be used alone or mixed together. Preferably, mixture of chloroform and methanol may be used. From the said extract, the isolation and the purification of the inventive sargachromenol can be performed by using the TLC for silica fractionation. However, the method for the isolation and the purification of the effective component are not limited to the above described (FIG. 1)
The sargachromenol of the present invention induces cytotoxicity and apoptosis of keratinocytes, thereby destroying and/or lysing keratinocytes at the skin area where the keratinocytes are hyperproliferating.
The sargachromenol of the present invention has cytotoxic effect on keratinocytes at the skin area where the keratinocytes are hyperproliferating. The keratinocytes hyperproliferate at the aged, photoaged or pigmented skin. Although not limited thereto, the pigmented skin refers to the skin where chloasma or dark spots are formed. In the examples that follow, cytotoxic effect on human keratinocytes treated by the sargachromenol of the present invention was confirmed through MTT analysis. The cytotoxic effect was concentration-dependent (see FIG. 2a).
Further, the sargachromenol of the present invention induces apoptosis of keratinocytes. In the examples that follow, inducement of apoptosis of human keratinocytes treated with sargachromenol was investigated through FACS and TUNEL analysis. Apoptosis effect was significantly higher than the human keratinocytes not treated by sargachromenol. The apoptosis effect increased as sargachromenol concentration and treatment time increased (see FIGS. 2b and 2c).
The sargachromenol of the present invention induces cell apoptosis by activating apoptosis-related factors. When human keratinocytes were treated with the sargachromenol of the present invention and activities of apoptosis-related factors were investigated, it was observed that caspase-8 and caspase-3 were cleaved by activation. Further, it was confirmed that the activities of apoptosis-related factors increased further when the concentration of the sargachromenol of the present invention was higher (see FIG. 3).
Cell apoptosis can be further promoted by , irradiating UV, in addition to treatment with sargachromenol.
In the examples that follow, apoptosis effect was investigated after treating human keratinocytes with sargachromenol followed by irradiation of UVB. Apoptosis effect was remarkably improved when the cells were irradiated with UV (see FIG. 4), and activities of apoptosis-related factors also increased further when UV was irradiated (see FIG. 5).
Accordingly, the present invention provides a method for destroying and/or lysing skin keratinocytes at the skin area where the keratinocytes are hyperproliferating, which is accomplished as the sargachromenol of the present invention induces cytotoxicity and apoptosis of skin keratinocytes by activating apoptosis-related factors. Further, the activity of destroying and/or lysing keratinocytes of the sargachromenol of the present invention is enhanced by irradiation of UV. Skin keratinocytes hyperproliferate because of skin aging, photoaging and pigmentation, resulting in, although not limited thereto, aged skin, wrinkles, chloasma and dark spots caused by UV in skin. Accordingly, the sargachromenol of the present invention, which has the activity of destroying and/or lysing keratinocytes, can be effectively used as keratinocyte lysing agent at the skin area where keratinocytes are hyperproliferating because of aging, photoaging and pigmentation. The keratinocyte lysing agent may also be used for skin peeling.
The present invention further provides a use of the sargachromenol represented by Chemical Formula I for the preparation of a keratinocyte lysing agent.
The present invention further provides a composition for lysing keratinocytes comprising the sargachromenol represented by Chemical Formula I and a dermatologically acceptable vehicle.
Keratinocytes, which account for over 95% of epidermal cells, produce the protein keratin and differentiate into basal cells, prickle cells and granular cells. Through turnover cycle, skin is continually generated and lost. But, when skin becomes aged or aged cells remain in the skin, the stratum corneum of the skin becomes thicker, resulting in skin disease such as wrinkles, chloasma, dark spots and the like.
A composition for lysing keratinocyte of this invention contains sargachromenol as an effective component, and may comprise a dermatologically acceptable vehicle and be prepared in the form of base makeup product (skin toner, cream, essence, face cleansing compositions such as cleansing foam, and cleansing water, pack, body oil), coloring cosmetics (foundation, lip stick, mascara, makeup base), haircare products (shampoo, rinse, hair conditioner, hair gel) and soap. As used herein, the term “dermatologically acceptable” refers to physiologically acceptable and when applied onto the skin or administered, generally does not cause allergic reactions or similar reactions thereto.
The vehicle of this invention may comprise, but not limited thereto, skin softener, skin infiltration enhancer, colorant, odorant, emulsifier, thickener, and solvent. In addition, it may comprise supplementary flavor, pigment, disinfectant, antioxidant, preservative, and humectant, and to improve physical property, it may comprise thickening agent, inorganic salts, synthetic polymers. For example, in case of manufacturing a face cleansing composition and a soap which containing sargachromenol of this invention, it may be easily prepared by adding sargachromenol to conventional cleansing composition or soap base. In case of manufacturing cream, it may be prepared by adding sargachromenol or the salt thereof to general oil-in-water cream base. In addition, it may comprise flavor, chelating agent, pigment, antioxidant, preservative and to improve physical property, it may comprise synthetic or natural materials such as protein, minerals, vitamins. In addition, composition for lysing keratinocyte of this invention may be prepared in the form of skin peeling reagent for chemical peeling.
The amount of sargachromenol in the composition for lysing keratinocyte of this invention may be 0.0001 to 50 w/w %, preferably 0.01 to 10 w/w %.
Hereinafter, the present invention will be described in detail by examples. It is to be understood well, however, that these examples are for illustrative purpose only and are not constructed to limit the scope of the present invention.